Brigham Young University BYU ScholarsArchive Theses and Dissertations 2016-06-01 Characterization of Five Brevibacillus Bacteriophages and Their Genomes Michael Allen Sheflo Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Sheflo, Michael Allen, "Characterization of Five Brevibacillus Bacteriophages and Their Genomes" (2016). Theses and Dissertations. 6059. https://scholarsarchive.byu.edu/etd/6059 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Characterization of Five Brevibacillus Bacteriophages and Their Genomes Michael Allen Sheflo A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Donald P. Breakwell, Chair Julianne H. Grose Sandra Hope Department of Microbiology and Molecular Biology Brigham Young University June 2016 Copyright © 2016 Michael Allen Sheflo All Rights Reserved ABSTRACT Characterization of Five Brevibacillus Bacteriophages and Their Genomes Michael Allen Sheflo Department of Microbiology and Molecular Biology, BYU Master of Science Brevibacillus laterosporus (B. laterosporus) is a pathogen difficult to distinguish from Paenibacillus larvae (P. larvae), and contributes to Colony Collapse Disorder (CCD) of honeybees. To develop a biocontrol agent to limit its presence, bacteriophages were isolated from Utah County soil samples and used to infect B. laterosporus isolated from Utah County honey and larvae samples. Since CCD is prevalent in Utah beehives, bacteriophage that infect and lyse B. laterosporus may be isolated and characterized. Pathogens were isolated from soil samples, and 16S rRNA gene tests initially identified the strains as P. larvae. Bacteriophages were isolated, purified, and amplified sufficiently to obtain images by electron microscope and genome sequencing by 454 pyrosequencing. Genomes were annotated with DNA Master, a Multiple Document Interface (MDI) program. Open reading frames (ORF’s) were compared to the National Center for Biotechnology Information’s (NCBI) database of primary biological sequence information via the Basic Local Alignment Search Tool (BLAST) algorithm. Later testing determined the pathogen to actually be B. laterosporus. Plaques demonstrated lytic activity, and electron microscopy revealed bacteriophages of the myoviridae family. The five sequenced genomes were composed of linear dsDNA ranging from 45,552 to 58,572 base pairs in length, 92 to 100 genes per genome, and a 38.10% to 41.44% range of G + C content. Discovering and describing new bacteriophages is a reasonably reproducible process and contributes to appreciating the diverse relationships between bacteriophage, bacteria, and eukaryota. Scientific facilitation of the bacteriophages role in limiting detrimental bacteria may contribute as an adjunctive therapy for CCD. Keywords: American Foulbrood, bacteriophage, Brevibacillus laterosporus, colony collapse disorder, European Foulbrood, genome, Paenibacillus larvae, Utah ACKNOWLEDGEMENTS I offer my sincere gratitude to Dr. Don Breakwell for his enthusiasm, optimism, and academic support throughout my time at BYU. I extend this gratitude to my graduate committee, Dr. Sandra Hope and Dr. Julianne Grose, for their ever-present wealth of knowledge and resources. I thank the research assistants from the Breakwell Laboratory from 2011 to 2013 for their assistance and creativity: Alicia Brighton, Josh Fisher, Adam Gardner, Jordan Jensen, Bryce Lunt, and Bryan Merrill. I thank my special research team for their support: Nic Ferguson, Ashley Kennedy, Carson Russell, and Kurt Williams. I thank the entire Utah County Beekeepers Association (UCBA) for supplying this endeavor with all necessary samples from their bee farms. I thank the BYU Department of Microbiology and Molecular Biology and the BYU College of Life Sciences for funding this research. I thank the Research Instrumentation Center (RIC) facility for access to their useful equipment, the BYU Sequencing Center (DNASC) for providing genomic information, and the BYU Microscopy Lab for all the amazing images. I thank the American Society of Microbiology for publishing the annotated genomes described here. I thank all the faithful tithe paying members of The Church of Jesus Christ of Latter-Day Saints for their sacrifice that makes possible the unique laboratory of learning that is Brigham Young University. Finally, I thank my parents, Larry and Mary, and my wife, Chelsie, for all their support. TABLE OF CONTENTS Title Page ......................................................................................................................................... i Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ...................................................................................................................................v List of Figures ................................................................................................................................ vi Abbreviations ................................................................................................................................ vii Chapter 1 – Introduction and Background .......................................................................................1 Chapter 2 – Materials and Methods ...............................................................................................16 Chapter 3 – Results of Isolation and Characterization...................................................................22 Chapter 4 – Discussion ..................................................................................................................29 Addendum ......................................................................................................................................36 Tables and Figures .........................................................................................................................40 Bibliography ..................................................................................................................................75 iv LIST OF TABLES Table 01 – Brevibacillus laterosporus isolated from Utah County honey ....................................40 Table 02 – Genome Summary of Five Brevibacillus Bacteriophage ............................................41 Table 03 – Detailed List of Jimmer 1 Genes .................................................................................42 Table 04 – Detailed List of Jimmer 2 Genes .................................................................................48 Table 05 – Detailed List of Emery Genes ......................................................................................54 Table 06 – Detailed List of Abouo Genes .....................................................................................60 Table 07 – Detailed List of Davies Genes .....................................................................................66 v LIST OF FIGURES Figure 01 – Plaque Morphologies of Five Brevibacillus Bacteriophage .......................................72 Figure 02 – Electron Microscope Images of Five Brevibacillus Bacteriophage ...........................73 Figure 03 – Restriction Endonuclease Digest of Five Brevibacillus Bacteriophage .....................74 vi ABBREVIATIONS AFB – American Foulbrood BLAST – Basic Local Alignment Tool BYU – Brigham Young University dsDNA – Double Stranded DNA LB – Lysis Broth ORF – Open Reading Frame PCR – Polymerase Chain Reaction Phage – Bacteriophage PL – Paenibacillus larvae TAE – tris acetate EDTA TE – tris EDTA TEM – transmission electron microscopy vii CHAPTER 1 – Introduction and Background The health and survival of honeybees is vital to sustaining our current world economy and ecosystem. Besides being the only insect that directly produces food consumed by people, honeybees also participate in nearly one third of all plant pollination worldwide (Johnson, 2010). Wild honeybees or commercially managed hives are used to pollinate fruit and nut trees not only throughout the United States, but globally (Morse, 2002). Truly, our ecosystem has developed to rely on the beneficial activities of honeybees, and our massive agricultural production has also benefited. Recently, honeybees have suffered an unusually high incidence of colony collapse, with an unprecedented decline of nearly half of all North American colonies (Johnson, 2010). Experts are still trying to determine if the decline is a novel phenomenon, but many have concluded that it is the result of a variety of causes. Such causes under investigation include weather fluctuations, human activities, and many well-studied bacterial, fungal, and viral diseases (Runckel, 2011). One cause is the bacterial disease, American Foulbrood (AFB) (Genersch, 2008; Eischen, 2005). Although AFB has been studied